Despite debate for more than two decades on the relative contribution of alloreactive versus autoreactive T cells in islet allograft destruction in autoimmune Type 1 diabetic (T1D) recipients, there has been a surprising paucity of basic studies that directly interrogate the types and specificities of T cells that actually infiltrate ad injure islet grafts in the autoimmune setting. This issue itself constitutes a glaring gap in curret information regarding the pathogenesis of islet transplant rejection in autoimmune recipients. Therefore, the general goal of this project is to determine the nature of T cells responsible for islet allograft destruction in the non-obese diabetic (NOD) pre-clinical model of T1D. Moreover, while it is clear that NOD mice are resistant to allograft tolerance, it is not at all clear what tpes of T cells are actually responsible for such tolerance-resistant graft rejection. Many previous studies have highlighted the role of memory T cells, inflammation, pathogen infection, and genetic resistance as general barriers to transplantation tolerance induction. This proposal sets out to explore the nature of spontaneous autoimmunity as another related endogenous barrier to allograft tolerance. That is, this project will also determine the nature of T cells (autoreactie and/or alloreactive) responsible for islet allograft recognition and, importantly, thos that resist tolerance-promoting therapies NOD mice. These general goals will be addressed through the following Specific Aims: Specific Aim 1: Determine primary T cell recognition pathway(s) that distinguish between the destruction of syngeneic versus allogeneic islet transplants in autoimmune NOD mice. This initial aim will test the hypothesis that CD4 T cells with autoimmune (islet-reactive) specificity comprise the primary component of cells targeting even MHC-unrelated islet allografts. This aim will attempt to either support or refute this primary hypothesis. Specific Aim 2: Determine the actual antigen specificity and T cells targeting islet transplants in NOD mice. This aim will attempt to support or refute the hypothesis that dual auto-/allo-reactive 'heterologous' T cells are specifically enriched in islet allografts. Specific im 3: Determine the nature of T cells responsible for 'tolerance resistant' islet allograft rejection n NOD mice. This Aim addresses the practical/translational issue of determining the nature of T cell reactivity that is refractory to tolerance-promoting therapies in the setting of autoimmune diabetes. There is a pressing need to develop novel and effective therapies to promote islet allograft survival in diabetic recipients. However, we believe that results from this project form primary and essential foundation for guiding such therapeutic design by clarifying the actual types of T cells that form key rate-limiting barriers to current treatment strategies.